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Home , Human coronavirus HKU1, Human metapneumovirus, Metapneumovirus

134 EU RO PE AN JOUR NAL OF MED I CAL RE SEARCH March 26, 2007

Eur J Med Res (2007) 12: 134-138 © I. Holzapfel Publishers 2007

TWO CASES OF SEVERE OBSTRUCTIVE ASSOCIATED WITH AN HKU1-LIKE

B. Kupfer2*, A. Simon1*, C. M. Jonassen4, S. Viazov3, V. Ditt3, R.-L. Tillmann2, A. Müller1, B. Matz2, O. Schildgen2

1Department of Pediatrics, 2Institute for Medical Microbiology, Immunology, and Parasitology, University of Bonn, 3Institute for Virology, Essen University Hospital, 4National Veterinary Institute, Norway

Abstract these pathogens could lead to mild to severe respirato- Background: During the last few years a number of ry tract (RTI) within all age groups. Despite previously undescribed , including human some reports from different parts of the world have , SARS, NL63 and described the occurrence of HKU1 coronavirus (Es- HKU1, and bocavirus, were identified in nasopharyn- per et al. 2006; Sloots et al. 2006; Vabret et al. 2006), geal samples from patients with signs of respiratory there is still little information about the , its dis- infections. These viruses may cause mild to life-threat- tribution, and its phylogeny. Here we describe the de- ening infections. tection of RNA from a new variant of HKU1 coron- Objectives: Nasopharyngeal samples from hospitalized avirus in two children hospitalized with respiratory pediatric patients with respiratory disease were tract infections in Germany. analysed for the presence of coronaviruses and other well known and newly identified respiratory viruses. PATIENTS AND METHODS Results: Two clinical cases of a severe obstructive pneumonia, which were associated with the presence CLINICAL SAMPLES AND PCR of RNA of a novel variant (subtype) of HKU1 coron- Nasopharyngeal aspirates were collected from hospi- avirus in the nasopharyngeal aspirates, were identified. talized pediatric patients between October 2003 and Discussion: The detection of a HKU1-like coronavirus May 2006 in the Department of Pediatrics of the Uni- in pediatric patients in the current study complement versity Hospital in Bonn, Germany. A total number of the most recent independent finding of similar or 296 samples were analysed for the presence of human closely related coronaviruses in patients with respira- coronaviruses. tory diseases in France (Vabret et al. 2006) and Nor- Therefore, RNA was extracted from nasopharyn- way (Jonassen et al., see accompanying manuscript). geal aspirates with the RNeasy protect kit (QIAGEN, These observations indicate a wide dissemination of Hilden, Germany) and subjected to a single-step RT- HKU1-like coronaviruses in Europe. PCR (QIAGEN, Hilden, Germany) using a set of primers designed on the basis of comparison of pol INTRODUCTION ORF1b sequences of known human coronaviruses. Basically, this approach was a modification of the Beside the commonly known viruses that cause respira- technique described by Stephensen et al. (1999) and tory tract infections in children like respiratory syncytial Moes et al. (2005). RT was carried out for 30 min at virus (RSV), , adenoviruses, and the human 42°C followed by a PCR with one initial step of Taq- coronaviruses OC43 and 229E, an increasing number Polymerase activation (95 °C for 15 min.), 35 cycles of of respiratory viruses were detected recently. These amplification (95 °C for 30 sec., 50 °C for 30 sec., pathogens include (van den 72 °C for 30 sec) and a final elongation step (72 °C for Hoogen et al. 2001), human bocavirus (Allander et al. 5 min). The primers used for the pan-coronavirus 2005) and three different coronaviruses. In 2003 one PCR were sv387as (5’ – TCA CAY TTW GGA TAR single outbreak of the SARS coronavirus evolved to a TCC CA), sv388s (5’ – ACT CAR ATR AAT CTT worldwide problem with high mortality rates of the in- AAR TAY GC), and sv389s (5’ – ACT CAA ATR fected patients (Drosten et al. 2003). Two other coron- AAT TTR AAR TAY GC) at final concentrations of aviruses, namely the human coronavirus NL-63 (van 0.6µM each. PCR-amplicons with a length of 251 der Hoek et al, 2004) also known as coronavirus NL basepairs were electrophoresed on an agarose gel and (Fouchier et al. 2004) and the human coronavirus visualized by ethidium bromide staining. HKU1 (Woo et al. 2005) have been identified recently. Samples were also screened for the presence of hu- The coronaviruses NL63 and HKU1 seem to be man metapneumovirus (HMPV) (Viazov et al. 2003; distributed worldwide and in contrast to SARS coron- Schildgen et al. 2004), RSV (Kupfer et al. 2006; Müller avirus, they occur more frequently. Infections with et al., submitted; Wilkesmann et al. 2006), viruses A and B (Müller et al., submitted for publica- tion; Wilkesmann et al. 2006), and bocavirus (Allander * The first two authors contributed equally to this paper. et al. 2005). March 26, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 135

SEQUENCING OF CORONAVIRAL PCR-AMPLICONS AND of an atypical pneumonia. His clinical picture was PHYLOGENETIC ANALYSIS characterized by a severe obstructive bronchitis, , wheezing, and tachypnoea (52 per minute). Amplicons were purified using the QIAquick PCR pu- Compared to patient 1 the clinical symptoms were less rification kit (Qiagen, Hilden, Germany) and subjected severe (no supplemental oxygen required), but severe to sequencing in both directions (BigDye Terminator enough to require a 7 days stay in hospital. At admis- DNA sequencing kit, Applied Biosystems, Darmstadt, sion the body temperature (36.9°C) was normal and Germany) as previously described (Schildgen et al. because of the severe respiratory symptoms he re- 2004). BlastN (http://www.ncbi.nlm.nih.gov/BLAST) ceived beta-mimetics, ipratropium bromide for 3 days, was used for comparison of the obtained sequences to and intravenous antibiotics for 4 days, despite the ab- nucleotide sequence databases. Viral sequences were sence of signs of an bacterial (CRP 3.6 aligned to the corresponding sequences of coron- mg/l, leukocyte count 6300/µl). avirus HKU1 (accession number: AY597011), SARS In nasopharyngeal washes from both patients, RNA coronavirus (AY515512), coronavirus OC43 from a novel HKU1-like coronavirus could be identi- (AY585229), coronavirus NL63 (AY567487), and a fied by RT-PCR. very recently described coronavirus strain (NO/0053/04; AM261821) using ClustalX version 1.8 PHYLOGENETIC ANALYSIS (Thompson et al. 1997). Phylogenetic trees of the newly derived sequences The sequences obtained from the direct were constructed by the neighbour-joining method sequencing of the coronavirus amplificons were ini- (Saitou & Nei, 1987). Reliability of the topology of tially submitted to a nucleotide-nucleotide blast the tree was evaluated by the bootstrap resampling (BlastN). This analysis revealed a similarity of 92% to method using 1000 replicates. The generated tree was the HKU1 group of coronaviruses. Most surprisingly, visualized with the program NJplot (Perrière et al. an exact match (100% identity) (Fig. 1A), and 99,5% 1996). identity, were found to coronaviruses NO/0053/04 and NO/0341/04, respectively, identified in Norway RESULTS from infants presenting respiratory tract infections in winter 2004/5 (Jonassen et al., submitted). Phyloge- In the present study 296 nasopharyngeal samples from netic analysis of the identified viruses (DE/0411 and hospitalized pediatric patients suffering from respira- DE/0415) from both patients together with tory symptoms were analysed by RT-PCR for coron- NO/0053/04 sequence revealed a aviruses. As controls, specimens from 15 healthy adult variant within the HKU1 cluster (Fig. 2). However, no volunteers were tested. differences between HKU1, DE/0411 and DE/0415 The most frequent pathogens detected were human were observed on the amino acid level (Fig. 1B). Of paramyxoviruses RSV and HMPV (manuscript in note, within the analysed amino acid sequence in this preparation). Coronaviruses were detected in five cas- portion of the viral replicase gene, there is only one es (1.7%). In three out of the five episodes of coron- substitution between HKU1 and OC43 (Fig. 1B). Phy- avirus-associated respiratory disease, the well charac- logenetic analysis showed higher divergence of the terized human coronavirus OC43 was identified by se- HKU1-like variants to the human pathogenic SARS quence analysis. Surprisingly, sequence analysis of coronavirus (branching off the root of the group II coronavirus PCR amplicons derived from the two re- coronaviruses) and coronavirus NL63 (group I coron- maining individuals revealed in both cases a novel avirus) on the nucleotide, as well as on the amino acid coronavirus variant. level (Fig. 1 and Fig. 2).

CLINICAL CASES DISCUSSION

The first patient with a coronavirus infection was a 16 During the last five years an increasing number of vi- months old boy admitted to the hospital with wheez- ral respiratory pathogens have been newly identified. ing, cough, (38.5°C), and a reduced general con- This includes three new coronaviruses, namely the dition. He was hospitalized for 14 days, and due to his SARS coronavirus (Drosten et al. 2003), the human reduced general condition and elevated inflammatory coronavirus NL63 (van der Hoek et al. 2004; Fouchier laboratory markers (CRP level 13.8 mg/L; leukocyte et al. 2004), and the coronavirus HKU1 (Woo et al. count 14.2 x 109/L) he received inhalative treatment 2005). Nevertheless, there is still a certain proportion with beta-mimetics, ipratropium bromide, budesonide of RTIs without an identified pathogen. Some RTIs in (7 days each) in addition with inhalative corticoids for our cohort were associated with human coronaviruses 6 days and systemic steroids for 3 days. No antibiotics but neither the SARS coronavirus nor the human were given since the chest radiography revealed no coronavirus NL63 could be detected in the nasopha- signs of a bacterial pneumonia. The child required ryngeal samples. The first reason for this observation supplemental oxygen for 4 days; the clinical severity is probably that the SARS outbreak was a rather single was 2 on the modified McIntosh scale (Wilkesmann et zoonotic event whereas other coronaviruses circulate al. 2006), probably due to a coinfection with HMPV. every year in the human population. Second, the coro- The second patient who suffered from a coron- navirus NL63 may be rare in our cohort of hospital- avirus infection (without evidence of other pathogens) ized patients. was an 18 months old boy who also showed symptoms In the present study and also in the work of 136 EUROPEAN JOURNAL OF MEDICAL RESEARCH March 26, 2007

A

B

G Fig. 1. A. Nucleotide alignments corresponding to the phylo- genetic tree. Sequences used for alignments were AY515512 (SARS), AY597011.2 (HKU1), AY585229 (OC43), AM261821 (NO/0053/04), and AY567487 (NL63) B. Align- ments of the predicted amino acid sequences within the poly- merase region from different coronaviruses. On the amino acid level no differences between the new variants NO/0053, DE/0411, DE/0415, and HKU1 were observed. Of note, the difference between HKU1 and OC43 within the analysed re- gion is only one single amino acid exchange.

F Fig. 2. Phylogenetic tree of human respiratory coronavirus- es. The novel variants NO/0053, DE/0411, and DE/0415 displayed a 92% homology to HKU1 on the nucleotide level and form a cluster of HKU1-like viruses, whilst reflecting a potential new genotype of HKU1. Sequences used for phylo- genetic analyses were AY515512 (SARS), AY597011.2 (HKU1), AY585229 (OC43), AM261821 (NO/0053/04), and AY567487 (NL63) March 26, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 137

Jonassen and colleagues (Jonassen et al., submitted) Escriou N, Grywna K, Kramme S, Manuguerra JC, the identification of a novel variant of coronaviruses Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Os- is described. These viruses were found in the nasopha- terhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe acute respirato- ryngeal secretions from children with acute respiratory ry syndrome. N Engl J Med. 2003; 348:1967-76. tract infections and are closely related to the HKU1 Esper F, Weibel C, Ferguson D, Landry ML, Kahn JS. Coro- coronavirus. However, the differences in the nu- navirus HKU1 infection in the United States. Emerg In- cleotide sequences compared to HKU1 were rather fect Dis. 2006; 12:775-9. high in this otherwise conserved part of the coron- Fouchier RA, Hartwig NG, Bestebroer TM, Niemeyer B, de avirus genome, even though none of the observed Jong JC, Simon JH, Osterhaus AD. A previously unde- substitutions was non-synonymous (i.e. 100% identity scribed coronavirus associated with respiratory disease in on the amino acid level between the novel viruses as humans. Proc Natl Acad Sci U S A. 2004; 101:6212-6. compared to previously published HKU1). One may Jonassen CM, Kofstad T, Dudman SG, Jonassen TØ. Detec- now speculate, however, that i) these viruses may rep- tion of different coronaviruses in Norwegian patients with respiratory symptoms, submitted to J. Clin. Virol. resent a novel coronavirus variant with different path- Kupfer B, Vehreschild J, Cornely O, Kaiser R, Plum G, Via- ogenic properties than the HKU-1 viruses identified zov S, Franzen C, Tillmann RL, Simon A, Müller A, and in Asia or ii) the newly identified HKU1-like variant Schildgen O: Severe pneumonia associated to new human reflects the seasonability of the HKU1 variants found bocavirus in a patient with a non- in different parts of the world at different times or III) (NHL); Emerg. Inf. 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Fatal pneumonia associated with human cal samples from patients with RTI for the occurrence metapneumovirus (HMPV) in a patient with myeloid of these newly identified coronaviruses, even if anoth- leukemia and adenocarcinoma in the lung. Eur J Med Res er pathogen was detected. In fact, in one of our two 12(4) (accepted) patients infected by the novel HKU1-like coronavirus Perrière G, Gouy M. WWW-Query: An on-line retrieval sys- variant no further pathogens were detected, whereas tem for biological sequence banks. Biochimie 1996; 78, the second patient was coinfected with HMPV. Fur- 364-369. thermore, the two Norwegian infants where the HKU- Saitou N, Nei M. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol 1 variants were identified were both infected with RSV Evol 1987; 4:406-25 (Jonassen et al., submitted). 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A newly discov- 138 EUROPEAN JOURNAL OF MEDICAL RESEARCH March 26, 2007

ered human pneumovirus isolated from young children Received: September 29, 2006 / Accepted: October 9, 2006 with respiratory tract disease. Nat Med. 2001; 7:719-24. van der Hoek L, Pyrc K, Jebbink MF, Vermeulen-Oost W, Address for correspondence: Berkhout RJ, Wolthers KC, Wertheim-van Dillen PM, Dr. rer. nat. Oliver Schildgen Kaandorp J, Spaargaren J, Berkhout B. Identification of a Institute for Medical Microbiology, Immunology, new human coronavirus. Nat Med. 2004; 10:368-73. and Parasitology Wilkesmann A, Schildgen O, Eis-Hubinger AM, Geikowski Department of Virology T, Glatzel T, Lentze MJ, Bode U, Simon A. Human University of Bonn metapneumovirus infections cause similar symptoms and Sigmund-Freud-Strasse 25 clinical severity as respiratory syncytial virus infections. 53105 Bonn Eur J Pediatr. 2006 Mar 23; [Epub ahead of print] Germany Tel.: +49-(0)228-28711697 Fax: +49-(0)228-28714433